Nectin-1: Synaptic processing and functions

Nectin-1：突触处理和功能

• 批准号: 7019434
• 负责人: HOWARD J. FEDEROFF
• 金额: $ 31.98万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-15 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7019434
• 关键词: adeno associated virus group; aspartic endopeptidases; cell adhesion molecules; cell line; developmental neurobiology; gene expression; hippocampus; immunoaffinity chromatography; laboratory mouse; laboratory rat; microarray technology; molecular chaperones; neural plasticity; neural transmission; neurogenetics; nuclear transport; point mutation; polymerase chain reaction; protein localization; protein structure function; proteolysis; synapses; synaptogenesis; transfection /expression vector; western blottings

DESCRIPTION (provided by applicant): Nectin-1 is a cell adhesion molecule localized the puncta adherentia junctions in synapses. Nectin-1 associates with other proteins, which collectively participate in the formation of neuronal synapses. We hypothesize that nectin-1-undergoes a regulated multi-step set of endoproteolytic cleavage events by several sheddases in an activity-dependent manner and that these events regulate synaptogenesis and contribute to synaptic plasticity. Our preliminary data indicate that nectin-1 undergoes ectodomain shedding by at least two sheddases that result in the production of two C-terminal fragments (CTFs). These CTFs are further cleaved intramembraneously by y-secretase and liberate the NE-ICD from the plasma membrane. The released NEICD translocates into the nucleus and where we postulate it induces gene expression. In Specific Aim 1, we will determine the secretase cleavage sites of nectin-1 by immunoaffinity purification, followed by Edman degradation sequencing. We will also investigate which genes are regulated by NE-ICD in hippocampal neurons by CodeLink Bioarrays. Then, using quantitative RT-PCR, ICC, and Western blotting we will confirm differentially expressed genes in neurons. The molecules that interact with NE-ICD will be identified by a yeast two-hybrid screen. Once interactors are identified, their biological function will be assayed by cellular and molecular approaches. In Specific Aim 2, we will investigate the biological role of BACE1 in nectin-1 processing. Initial experiments indicate that BACE1 associates with and participates in the shedding of nectin-1. We will investigate how disruption of nectin-1 shedding, through loss of BACE1 function, affects synapse formation and synaptic plasticity by ICC, Western blotting and-vesicle recycling assays. Our preliminary data indicate that two nectin-1 point mutations, T310A and Y311A, are refractory to BACE1 cleavage and can /ra".y-dominantly interfere with processing of endogenous nectin-1. We will examine how these point mutants affect the synapse formation and synaptic function by transduction of hippocampal neurons and in vivo adult hippocampus with recombinant adeno-associated viral vectors. We will quantitatively measure the changes in synaptic markers, synapse morphology, and size by ICC, live cell imaging and synaptic activity. Subsequently, we examine whether expression of trans-dominant nectin-1 mutants will affect hippocampal dependent learning

描述（由申请人提供）：nectin-1是一种细胞粘附分子，将突触中的点粘附连接处定位。 Nectin-1与其他蛋白质相关，共同参与了神经元突触的形成。我们假设nectin-1-在go中通过几个sheddass以活性依赖性的方式通过多个sheddass的多个多蛋白溶解裂解事件，这些事件调节突触发生并有助于突触可塑性。我们的初步数据表明，Nectin-1通过至少两个sheddass进行过域域的脱落，这些酶产生了两个C末端片段（CTF）。这些CTF通过Y-分泌酶进一步裂解，并从质膜中释放NE-ICD。释放的NEICD易位到核中，我们假设其诱导基因表达。在特定的目标1中，我们将通过免疫亲和力纯化确定Nectin-1的泌尿酶裂解位点，然后进行Edman降解测序。我们还将研究哪些基因由CodeLink生物阵列中海马神经元中的NE-ICD调节。然后，使用定量RT-PCR，ICC和Western印迹我们将确认神经元中差异表达的基因。与NE-ICD相互作用的分子将通过酵母两杂化筛选鉴定。一旦确定了相互作用的人，将通过细胞和分子方法来测定它们的生物学功能。在特定目标2中，我们将研究BACE1在Nectin-1加工中的生物学作用。最初的实验表明，BACE1与Nectin-1的脱落并参与。我们将研究Nectin-1脱落的破坏是如何通过ICC，Western blotting and-vesicle回收测定法影响突触形成和突触可塑性。我们的初步数据表明，两个nectin-1点突变T310a和Y311a对Bace1裂解具有难体，can /can /can /ra ".y。主要干扰内源性的Nectoil nectoil nectoil nectoil nectoil nectoil nectoil nection-1。我们将研究这些点突变体如何影响突触的形成，并与诱导型成年型成年型和Inipbins hippins Neuruls to traptiant and viv型和viv型，并在Recombl neurs上进行了hippin neurys和Viv的突触功能。与腺相关的病毒载体。